Miller A L, Kress B C, Stein R, Kinnon C, Kern H, Schneider J A, Harms E
J Biol Chem. 1981 Sep 10;256(17):9352-62.
Using a combination of differential centrifugation and free flow electrophoresis (Harms, E., Kern, H., and Schneider, J. A. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 6139-6143) a single population of highly purified lysosomes was obtained from normal, I-cell disease type 1, and I-cell disease type 2 cultured fibroblasts. Our findings indicate that most of the residual acid hydrolase activities remaining within the I-cell fibroblasts are localized in the lysosomes, analogous to normal cells. Characterization of the carbohydrate-dependent properties of the lysosomal N-acetyl-beta-D-hexosaminidase revealed that the I-cell and normal enzymes do not contain a significant proportion of neuraminidase-susceptible sialic acid residues, interact poorly with the beta-galactose-specific lectin Ricinus communis and are highly sensitive to endohexosaminidase H treatment, indicating that the oligosaccharide units of both the I-cell and normal lysosomal N-acetyl-beta-D-hexosaminidase are predominantly of the high mannose type. The I-cell and normal lysosomal N-acetyl-beta-D-hexosaminidase, however, differed in their endocytotic properties. In contrast to the high rate of endocytosis of the normal lysosomal enzyme (7.8%/mg/h), the I-cell type 1 lysosomal enzyme failed to be endocytosed into Sandhoff cells indicating an absent or altered phosphohexyl recognition marker on the I-cell enzyme. Examination of the normal extracellular N-acetyl-beta-D-hexosaminidase revealed the presence of predominantly high mannose-type oligosaccharide units, similar to the corresponding lysosomal enzyme, although properties typical of complex-type oligosaccharide chains were also evident. In contrast, the secreted I-cell enzyme revealed the presence of oligosaccharide units predominantly of the complex type indicating that the I-cell N-acetyl-beta-D-hexosaminidase has had high mannose-type oligosaccharide chains modified to complex-type probably in the Golgi or GERL region prior to secretion from the cell.
通过差速离心和自由流动电泳相结合的方法（哈姆斯，E.，克恩，H.，和施耐德，J. A.（1980年）《美国国家科学院院刊》77，6139 - 6143），从正常、1型I细胞病和2型I细胞病培养的成纤维细胞中获得了单一群体的高度纯化的溶酶体。我们的研究结果表明，I细胞成纤维细胞内剩余的大部分酸性水解酶活性定位于溶酶体中，这与正常细胞类似。对溶酶体N - 乙酰 - β - D - 己糖胺酶的碳水化合物依赖性特性的表征显示，I细胞和正常细胞的酶不含有显著比例的对神经氨酸酶敏感的唾液酸残基，与β - 半乳糖特异性凝集素蓖麻毒素相互作用不佳，并且对内切己糖胺酶H处理高度敏感，这表明I细胞和正常溶酶体N - 乙酰 - β - D - 己糖胺酶的寡糖单元主要是高甘露糖型。然而，I细胞和正常溶酶体N - 乙酰 - β - D - 己糖胺酶在其胞吞特性方面存在差异。与正常溶酶体酶的高胞吞率（7.8%/mg/h）相反，1型I细胞溶酶体酶未能被内吞入桑德霍夫细胞，这表明I细胞酶上不存在或改变了磷酸己基识别标记。对正常细胞外N - 乙酰 - β - D - 己糖胺酶的检查显示，主要存在高甘露糖型寡糖单元，类似于相应的溶酶体酶，尽管也明显存在复杂型寡糖链的典型特性。相比之下，分泌的I细胞酶显示主要存在复杂型寡糖单元，这表明I细胞N - 乙酰 - β - D - 己糖胺酶的高甘露糖型寡糖链可能在从细胞分泌之前在高尔基体或GERL区域被修饰为复杂型。
